SIGIRR胞内区酵母双杂交诱饵质粒的构建及自激活作用检测  

Construction of bait plasmid containing SIGIRR cytoplasmic tail and detection of self-activation in yeast two hybrid system

作　　者：陈旭昕[1] 冯华松[1] 段蕴铀[1] 吴学玲[2] 钱桂生[2] 

机构地区：[1]中国人民解放军海军总医院呼吸内科,北京100037 [2]第三军医大学新桥医院呼吸内科,重庆400037

出　　处：《重庆医学》2014年第10期1164-1167,共4页Chongqing medicine

基　　金：国家自然科学基金资助项目(30600272)

摘　　要：目的：构建单免疫球蛋白白细胞介素-1受体相关蛋白（SIGIRR）胞内区酵母双杂交诱饵质粒，并检测其是否存在自激活作用。方法聚合酶链反应（PCR）扩增人SIGIRR胞内区基因片段（480～1230 bp），并将此基因片段重组入pSos载体中，构建诱饵质粒pSos-SIGIRR ，经酶切及测序鉴定构建正确后，将重组质粒与对照质粒共转化感受态酵母菌cdc25H ，接种于25℃SD/Glucose（-UL）和SD/Galactose（-UL）平板以及37℃ SD/Glucose（-UL）和SD/Galactose（-UL）平板上，连续观察6 d酵母菌生长状况；用Western blot法检测目的蛋白表达情况。结果正确构建了人SIGIRR胞内区酵母双杂交诱饵质粒pSos-SIGIRR ， pSos-SIGIRR在酵母双杂交系统中无自激活及毒性作用。Western blot结果显示，目的蛋白以170＆#215;103的融合蛋白形式表达。结论诱饵质粒pSos-SIGIRR可应用于酵母双杂交系统中，为在人肺互补脱氧核糖核酸（cDNA ）文库中寻找与SIGIRR相互作用蛋白奠定了重要基础。Objective To construct the bait plasmid of pSos-single immunoglobin IL-1 receptor related protein （SIGIRR） in Cy-toTrap yeast two hybrid system ,and to test its self-activation .Methods The cDNA fragments of SIGIRR（480 -1 230 bp） were amplified from pReceiver-LV19-SIGIRR and ligated into the bait plasmid pSos to generate the plasmid pSos-SIGIRR .The pSos-SI-GIRR was identified by DNA sequencing and dual-site endonuclease digestion .Then the recombinant plasmid and control plasmid were introduced into the yeast cell cdc25H .The transformants were inoculated on plates of 25 ℃ /SD/Glucose（-UL） ,25 ℃/SD/Ga-lactose（-UL） ,37 ℃ /SD/Glucose（-UL） and 37 ℃ /SD/Galactose ,respectively and the proliferation ability of transformant was ob-served for 6 d .The Western blot was adopted to detect the expression of target protein .Results The pSos-SIGIRR vector was cor-rectly constructed and proved of no self-activation and toxic action .The Western blot showed that the target protein was expressed in a form of fusion protein of 170KD .Conclusion The bait plasmid containing SIGIRR cytoplasmic tail can be applied to the yeast two-hybrid system and lays the important foundation for seeking the interacting protein with SIGRR from the human lung cDNA li-brary in .

关 键 词：免疫球蛋白白细胞介素1受体相关蛋白 双杂交系统技术 诱饵质粒 自激活 

分 类 号：R346[医药卫生—基础医学]